Microrna-9 Promotes Proliferation of Leukemia Cells in Adult CD34 Positive Acute Myeloid Leukemia with Normal Karyotype By Down-Regulation of Hes1

Abstract Acute myeloid leukemia (AML) is a group of heterogeneous hematopoietic malignancies sustained by a small population of leukemic stem cells (LSCs) that can resist treatment and act as barriers to cure. Previously, we observed that Hes1 and p21 expression was down-regulated in AML cell lines compared to that of normal bone marrow mononuclear cells. However, the activation status of Hes1-p21 pathway and its regulation in LSCs as well as normal hematopoietic stem cells (HSCs) in AML has not been elucidated. In this study, the Hes1-p21 pathway in LSCs and leukemic progenitors (LPs) was studied in adult CD34+ AML with normal karyotype and no genetic mutations and the upstream miRNA regulators were screened. Our results showed that the level of either Hes1 or p21 was lower in LSCs or LPs than that of HSCs whereas the level of miR-9 was higher in LSCs or LPs than HSCs. An inverse correlation was observed in the expression of Hes1 and miR-9. Furthermore, we validated miR-9 as one of the regulators of Hes1 by reporter gene analysis. Knockdown of miR-9 by lentivirus infection suppressed the proliferation of AML cells by the induction of G0 arrest and apoptosis in vitro. Moreover, knockdown of miR-9 resulted in decreased circulating leukemic cell counts in peripheral blood and bone marrow, attenuated splenomegaly, and prolonged survival in a xenotransplant mouse model. Our results indicate that the miR-9-Hes1-p21 pathway plays an important role in supporting AML cell growth and survival, and that miR-9 has a potential to be a therapeutic target for suppressing AML. Disclosures No relevant conflicts of interest to declare.

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Gpr44 Mediates the Selenium-Dependent Anti-Leukemic Effect in Acute Myeloid Leukemia

Current Developments in Nutrition ◽

10.1093/cdn/nzaa067_062 ◽

2020 ◽

Vol 4 (Supplement_2) ◽

pp. 1835-1835

Author(s):

Fenghua Qian ◽

Diwakar Tukaramrao ◽

Jiayan Zhou ◽

Nicole Palmiero ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Murine Model ◽

Myeloid Leukemia ◽

Hematopoietic Stem ◽

Cell Counts ◽

Pparγ Agonist ◽

Acute Myeloid

Abstract Objectives The relapse of acute myeloid leukemia (AML) remains a significant concern due to persistent leukemia stem cells (LSCs) that are not targeted by existing therapies. LSCs show sensitivity to endogenous cyclopentenone prostaglandin J (CyPG) metabolites that are increased by dietary trace element selenium (Se), which is significantly decreased in AML patients. We investigated the anti-leukemic effect of Se supplementation in AML via mechanisms involving the activation of the membrane-bound G-protein coupled receptor 44 (Gpr44) and the intracellular receptor, peroxisome proliferator-activated receptor gamma (PPARγ), by endogenous CyPGs. Methods A murine model of AML generated by transplantation of hematopoietic stem cells (HSCs- WT or Gpr44−/−) expressing human MLL-AF9 fusion oncoprotein, in the following experiments: To investigate the effect of Se supplementation on the outcome of AML, donor mice were maintained on either Se-adequate (Se-A; 0.08–0.1 ppm Se) or Se-supplemented (Se-S; 0.4 ppm Se) diets. Complete cell counts in peripheral blood were analyzed by hemavet. LSCs in bone marrow and spleen were analyzed by flow cytometry. To determine the role of Gpr44 activation in AML, mice were treated with Gpr44 agonists, CyPGs. LSCs in bone marrow and spleen were analyzed. Mice transplanted with Gpr44−/- AML cells were compared with mice transplanted with wild type AML cells and the progression of the disease was followed as above. To determine the role of PPARγ activation in AML, PPARγ agonist (Rosiglitazone, 6 mg/kg, i.p, 14 d) and antagonist (GW9662, 1 mg/kg, i.p. once every other day, 7 injections) were applied to Se-S mice transplanted with Gpr44−/- AML cells and disease progression was followed. Results Se supplementation at supraphysiological levels alleviated the disease via the elimination of LSCs in a murine model of AML. CyPGs induced by Se supplementation mediate the apoptosis in LSCs via the activation of Gpr44 and PPARγ. Conclusions Endogenous CyPGs produced upon supplementation with Se at supraphysiological levels improved the outcome of AML by targeting LSCs to apoptosis via the activation of two receptors, Gpr44 and PPARg. Funding Sources NIH DK 07,7152; CA 175,576; CA 162,665. Office of Dietary Supplements, USDA Hatch funds PEN04605, Accession # 1,010,021 (KSP, RFP).

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7-Ketocholesterol Promotes Oxiapoptophagy in Bone Marrow Mesenchymal Stem Cell from Patients with Acute Myeloid Leukemia

Cells ◽

10.3390/cells8050482 ◽

2019 ◽

Vol 8 (5) ◽

pp. 482 ◽

Cited By ~ 5

Author(s):

Jessica Liliane Paz ◽

Debora Levy ◽

Beatriz Araujo Oliveira ◽

Thatiana Correia de Melo ◽

Fabio Alessandro de Freitas ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Cholesterol Oxidation ◽

Specific Cell ◽

Hematopoietic Stem ◽

Shh Pathway ◽

Number Of Cells ◽

Acute Myeloid

7-Ketocholesterol (7-KC) is a cholesterol oxidation product with several biological functions. 7-KC has the capacity to cause cell death depending on the concentration and specific cell type. Mesenchymal stem cells (MSCs) are multipotent cells with the ability to differentiate into various types of cells, such as osteoblasts and adipocytes, among others. MSCs contribute to the development of a suitable niche for hematopoietic stem cells, and are involved in the development of diseases, such as leukemia, to a yet unknown extent. Here, we describe the effect of 7-KC on the death of bone marrow MSCs from patients with acute myeloid leukemia (LMSCs). LMSCs were less susceptible to the death-promoting effect of 7-KC than other cell types. 7-KC exposure triggered the extrinsic pathway of apoptosis with an increase in activated caspase-8 and caspase-3 activity. Mechanisms other than caspase-dependent pathways were involved. 7-KC increased ROS generation by LMSCs, which was related to decreased cell viability. 7-KC also led to disruption of the cytoskeleton of LMSCs, increased the number of cells in S phase, and decreased the number of cells in the G1/S transition. Autophagosome accumulation was also observed. 7-KC downregulated the SHh protein in LMSCs but did not change the expression of SMO. In conclusion, oxiapoptophagy (OXIdative stress + APOPTOsis + autophagy) seems to be activated by 7-KC in LMSCs. More studies are needed to better understand the role of 7-KC in the death of LMSCs and the possible effects on the SHh pathway.

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TIM-3 Is a Promising Target to Eradicate Acute Myeloid Leukemia Stem Cells Sparing Normal Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v118.21.559.559 ◽

2011 ◽

Vol 118 (21) ◽

pp. 559-559

Author(s):

Toshihiro Miyamoto ◽

Yoshikane Kikushige ◽

Takahiro Shima ◽

Koichi Akashi

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Hematopoietic Stem Cells ◽

Myeloid Leukemia ◽

Leukemia Cell ◽

Cytotoxic Activities ◽

Antibody Treatment ◽

Hematopoietic Stem ◽

Acute Myeloid

Abstract Abstract 559 Acute myeloid leukemia (AML) originates from self-renewing leukemic stem cells (LSCs), an ultimate therapeutic target for permanent cure. To selectively kill AML LSCs sparing normal hematopoietic stem cells (HSCs), one of the most practical approaches is to target the AML LSCs-specific surface or functionally indispensable molecules. Based on differential transcriptome analysis of prospectively-purified CD34+CD38− LSCs from AML patient samples and normal HSCs, we found that T-cell immunoglobulin mucin-3 (TIM-3) was highly expressed in AML LSCs but not in normal HSCs (Kikushige et al., Cell Stem Cell, 2010). In normal hematopoiesis, TIM-3 is mainly expressed in mature monocytes and a fraction of NK cells, but not in granulocytes, T cells or B cells. In the bone marrow, TIM-3 is expressed only in a fraction of granulocyte/macrophage progenitors (GMPs) at a low level, but not in HSCs, common myeloid progenitors, or megakaryocyte/erythrocyte progenitors. In contrast, in human AML, TIM-3 was expressed on cell surface of the vast majority of CD34+CD38− LSCs and CD34+CD38+ leukemic progenitors in AML of most FAB types, except for acute promyelocytic leukemia (M3). FACS-sorted TIM-3+ but not TIM-3− AML cells reconstituted human AML in the immunodeficient mice, indicating that the TIM-3+ population contains most of functional LSCs. To selectively eradicate TIM-3-expressing AML LSCs, we established an anti-human TIM-3 mouse IgG2a antibody, ATIK2a, possessing antibody-dependent cellular cytotoxic and complement-dependent cytotoxic activities in leukemia cell lines transfected with TIM-3. We first tested the effect of ATIK2a treatment on reconstitution of normal HSCs in a xenograft model. ATIK2a was intraperitoneally injected to the mice once a week after 12 hours of transplantation of human CD34+ cells. Injection of ATIK2a did not affect reconstitution of normal human hematopoiesis except removing TIM-3-expressing mature monocytes. In contrast, injection of TIM-3 to the mice transplanted with human AML samples markedly reduced leukemic repopulation. In some mice transplanted with AML bone marrow, only normal hematopoiesis was reconstituted after anti-TIM-3 antibody treatment, suggesting that the antibody selectively killed AML cells, sparing residual normal HSCs. To further test the inhibitory effect of ATIK2a on established human AML, eight weeks after transplantation of human AML cells, engraftment of human AML cells was confirmed by blood sampling and thereafter ATIK2a was injected to these mice. In all cases tested, ATIK2a treatment significantly reduced human TIM-3+ AML fraction as well as the CD34+CD38− LSCs fraction. In addition, to verify the anti-AML LSCs effect of ATIK2a treatment, human CD45+AML cells from the primary recipients were re-transplanted into secondary recipients. All mice transplanted from primary recipients treated with control IgG developed AML, whereas none of mice transplanted with cells from ATIK2a-treated primary recipients developed AML, suggesting that functional LSCs were effectively eliminated by ATIK2a treatment in primary recipients. Thus, TIM-3 is a promising surface molecule to target AML LSCs. Our experiments strongly suggest that targeting this molecule by monoclonal antibody treatment is a practical approach to eradicate human AML. Disclosures: No relevant conflicts of interest to declare.

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CXCR4 Invalidation Limits the MLL-ENL Induced Leucemogenesis in Vivo

Blood ◽

10.1182/blood.v120.21.4089.4089 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4089-4089

Author(s):

Yanyan Zhang ◽

Hadjer Abdelouahab ◽

Aline Betems ◽

Monika Wittner ◽

William Vainchenker ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Survival Time ◽

Median Survival ◽

Median Survival Time ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Hematopoietic Stem ◽

Acute Myeloid

Abstract Abstract 4089 The receptor CXCR4 and its ligand SDF-1 play major physiological roles especially on hematopoietic stem cells homing and retention. Many studies have implicated CXCR4 in the invasion by tumor cells of organs that produce SDF-1. In acute myeloid leukemia, the physiological role of CXCR4 is not fully understood. We used retrovirus to express MLL-ENL oncogene in CXCR4+/+ and CXCR4−/− hematopoietic primitive cells (Lin- isolated from fetal liver) and showed that CXCR4 is dispensable for generation of immortalized colonies in vitro. To determine CXCR4 function in vivo, CXCR4+/+ and CXCR4−/− transformed cells were transplanted into lethally irradiated mice. Whatever their phenotype, the recipient developed a myelo-monocytique leukemia characterized by their expression of Gr-1 and Mac-1. As expected, all recipients of MLL-ENL transduced CXCR4+/+ cells were moribund within 35 to 80 days post transplant (median survival time: 50 days). Strikingly, recipients of MLL-ENL transduced CXCR4−/− cells showed significantly increased lifespan, with a median survival time of 90 days. The cellularity of the peripheral blood of recipients of MLL-ENL transduced cells displayed considerable increases over time although this increase was much lower in CXCR4−/− than in CXCR4+/+ chimera. Bone marrow of MLL-ENL transduced CXCR4−/− chimera had moderately decreased numbers of mononuclear cells. There were important numbers of leukemic CD45.2+/Gr1+/Mac1+/c-kit+ cells in spleen from MLL-ENL CXCR4+/+ chimera which suggested that CXCR4 is important for leukemic progenitors cells retention in the bone marrow and especially in the spleen. The homing capacity of transduced CXCR4+/+ cells is comparable to the CXCR4−/− cells. Finally, more DNA damages were found in the BM cells of MLL-ENL CXCR4−/− chimera. All these results were confirmed by treating of MLL-ENL CXCR4+/+ chimera with CXCR4 inhibitor (TN140). These results demonstrated that in absence of CXCR4, the cells transduced by oncogene MLL-ENL are capable of generating leukemia in the recipients. However, mice transplanted with MLL-ENL transduced CXCR4−/− FL cells developed acute myeloid leukemia with reduced aggressiveness and organ infiltration, which is associated with induced differentiation and DNA instability. These results indicated that the MLL-ENL progenitors are dependent on CXCR4 for their maintenance in the BM and spleen suggesting that CXCR4 inhibitors might have potential therapeutic applications. Disclosures: No relevant conflicts of interest to declare.

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Inhibition of HDAC8 Reactivates p53 and Abrogates Leukemia Stem Cell Activity in CBFβ-SMMHC Associated Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v124.21.363.363 ◽

2014 ◽

Vol 124 (21) ◽

pp. 363-363

Author(s):

Jing Qi ◽

Qi Cai ◽

Sandeep Singh ◽

Ling Li ◽

Hongjun Liu ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Cell Activity ◽

Disease Free Survival ◽

Hematopoietic Stem ◽

The Impact ◽

Acute Myeloid

Abstract The inv(16)-created CBFβ-SMMHC fusion protein inhibits differentiation of hematopoietic stem and progenitor cells (HSPCs) and creates pre-leukemic populations predisposed to acute myeloid leukemia (AML) transformation. However, the molecular mechanism underlying the leukemogenic function of CBFβ-SMMHC has been elusive. Given the low TP53 mutation rate in AML, alternative mechanisms disrupting p53 function are expected. We showed thatCBFβ-SMMHC impairs p53 acetylation and p53 target gene activation through formation of an aberrant protein complex with p53 and HDAC8 (Blood, 120: A772; 122(21): 224). We now show that CBFβ-SMMHC binds to p53 and HDAC8 independently through distinct regions and that HDAC8 mediates the deacetylation of p53 associated with CBFβ-SMMHC. In addition, we generated mice carrying a floxed Hdac8 (Hdac8f) allele and crossed with Cbfb56M/+/Mx1-Cre (Kuo YH et al, Cancer Cell 2006). Deletion of Hdac8 signifiacntly (p<0.0001) reduced the incidence of AML and prolonged disease-free survival. Pharmacologic inhibition of HDAC8 activity with HDAC8-selective inhibitors (HDAC8i) reactivates p53 and selectively induces apoptosis of inv(16)+ AML CD34+ cells while sparing normal HSPCs. To test the effect of HDAC8i on LSC engraftment and leukemia-initiating capacity, we generated Cbfb56M/+/Mx1-Cre mice with a Cre-reporter line expressing tdTomato fluorescence protein following Cre-mediated recombination. AML cells (dTomato+/cKit+) treated with HDAC8i (22d) ex vivo showed reduced engraftment (p=0.025) and enhanced survival (p=0.025) in transplanted mice. To examine whether HDAC8i 22d treatment affects the engraftment capacity on surviving cells, we transplanted equal number (2 x 106) of AML cells treated with either 22d or vehicle in another cohort of mice (n=4). We show that HDAC8i 22d treatment reduced the engraftment of dTomato+/cKit+ AML cells and enhanced survival, suggesting that the engraftment capacity is altered in addition to reducing AML cell survival. We next performed preclinical studies to determine the efficacy of in vivo administration of HDAC8i 22d. AML transplanted mice were randomized into two groups, one group treated with vehicle and the other treated with HDAC8i 22d for 2 weeks. Flow cytometry analysis revealed significantly reduced frequency (p=0.0097) and number (p=0.0101) of dTomato+/cKit+ AML cells in the bone marrow and spleen of 22d treated mice compared to vehicle treated group. To further assess the impact on LSC activity, we transplanted bone marrow cells from these treated mice into secondary recipients and analyzed for AML engraftment. Significant reduction in the frequency (p<0.0001) and the number (p=0.0006) of dTomato+/cKit+ AML cells was observed in the bone marrow and spleen. Furthermore, HDAC8i 22d treated transplants showed no signs of leukemia while vehicle treated transplants are moribund with aggressive AML. These results indicate that HDAC8 inhibition by 22d treatment effectively eliminates engraftment and leukemia-initiating capacity of AML LSCs. In conclusion, our studies identify a novel post-translational p53-inactivating mechanism and demonstrate selective HDAC8 inhibition as a promising approach to target inv(16)+ AML LSCs. Disclosures No relevant conflicts of interest to declare.

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CXCR4 Antagonists as Stem Cell Mobilizers and Therapy Sensitizers for Acute Myeloid Leukemia and Glioblastoma?

Biology ◽

10.3390/biology9020031 ◽

2020 ◽

Vol 9 (2) ◽

pp. 31 ◽

Cited By ~ 3

Author(s):

Vashendriya V.V. Hira ◽

Cornelis J.F. Van Noorden ◽

Remco J. Molenaar

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Stem Cell ◽

Myeloid Leukemia ◽

Patient Survival ◽

Hematopoietic Stem ◽

Poor Patient ◽

Acute Myeloid ◽

Poor Patient Survival

Glioblastoma is the most aggressive and malignant primary brain tumor in adults and has a poor patient survival of only 20 months after diagnosis. This poor patient survival is at least partly caused by glioblastoma stem cells (GSCs), which are slowly-dividing and therefore therapy-resistant. GSCs are localized in protective hypoxic peri-arteriolar niches where these aforementioned stemness properties are maintained. We previously showed that hypoxic peri-arteriolar GSC niches in human glioblastoma are functionally similar to hypoxic peri-arteriolar hematopoietic stem cell (HSC) niches in human bone marrow. GSCs and HSCs express the receptor C-X-C receptor type 4 (CXCR4), which binds to the chemoattractant stromal-derived factor-1α (SDF-1α), which is highly expressed in GSC niches in glioblastoma and HSC niches in bone marrow. This receptor–ligand interaction retains the GSCs/HSCs in their niches and thereby maintains their slowly-dividing state. In acute myeloid leukemia (AML), leukemic cells use the SDF-1α–CXCR4 interaction to migrate to HSC niches and become slowly-dividing and therapy-resistant leukemic stem cells (LSCs). In this communication, we aim to elucidate how disruption of the SDF-1α–CXCR4 interaction using the FDA-approved CXCR4 inhibitor plerixafor (AMD3100) may be used to force slowly-dividing cancer stem cells out of their niches in glioblastoma and AML. Ultimately, this strategy aims to induce GSC and LSC differentiation and their sensitization to therapy.

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The leukemic stem cell niche: current concepts and therapeutic opportunities

Blood ◽

10.1182/blood-2009-01-202606 ◽

2009 ◽

Vol 114 (6) ◽

pp. 1150-1157 ◽

Cited By ~ 316

Author(s):

Steven W. Lane ◽

David T. Scadden ◽

D. Gary Gilliland

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Bone Marrow Microenvironment ◽

Self Renewal ◽

Hematopoietic Stem ◽

Rational Development ◽

Cell Niche ◽

Acute Myeloid

Abstract The genetic events that contribute to the pathogenesis of acute myeloid leukemia are among the best characterized of all human malignancies. However, with notable exceptions such as acute promyelocytic leukemia, significant improvements in outcome based on these insights have not been forthcoming. Acute myeloid leukemia is a paradigm of cancer stem (or leukemia initiating) cells with hierarchy analogous to that seen in hematopoiesis. Normal hematopoiesis requires complex bidirectional interactions between the bone marrow microenvironment (or niche) and hematopoietic stem cells (HSCs). These interactions are critical for the maintenance of normal HSC quiescence and perturbations can influence HSC self-renewal. Leukemia stem cells (LSCs), which also possess limitless self-renewal, may hijack these homeostatic mechanisms, take refuge within the sanctuary of the niche during chemotherapy, and consequently contribute to eventual disease relapse. We will discuss the emerging evidence supporting the importance of the bone marrow microenvironment in LSC survival and consider the physiologic interactions of HSCs and the niche that inform our understanding of microenvironment support of LSCs. Finally, we will discuss approaches for the rational development of therapies that target the microenvironment.

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Results of Hematopoietic Stem Cell Transplantation for Acute Myeloid Leukemia of the FAB M0 Subtype in First Complete Remission: A Survey of the Acute Leukemia Working Party of the European Bone Marrow Transplant Group (EBMT).

Blood ◽

10.1182/blood.v104.11.5131.5131 ◽

2004 ◽

Vol 104 (11) ◽

pp. 5131-5131

Author(s):

Andree-Laure Herr ◽

Myriam Labopin ◽

Rosy Reiffers ◽

Donald Bunjes ◽

Didier Blaise ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Stem Cell ◽

Hematopoietic Stem Cell Transplantation ◽

Complete Remission ◽

Myeloid Leukemia ◽

Hematopoietic Stem ◽

First Complete Remission ◽

Acute Myeloid

Abstract Hematopoietic stem cell transplantation (HSCT) is a potentially curative treatment for patients with acute myeloid leukemia (AML). AML of the FAB M0 subtype is rare, often associated with a complex karyotype and a poor prognosis. Results of HSCT for this AML subtype have never been reported separately from other subtypes. We did a survey of the results of 274 HSCT in adults with M0 AML in first complete remission (CR1), performed in EBMT centres since January 1990 until 2002. One hundred fifty patients were transplanted with an HLA identical donor (HLA-id), 30 with an HLA-matched unrelated donor (MUD) and 94 received an autologous transplant (auto). The median age was 45 years (16–71), the median interval from diagnosis to HSCT was 4 months for HLA-id, 6 months for MUD and 5 months for auto HSCT. The median follow-up time (range) was 20 months (1–109), 12 (2–53) and 10 months (1–96) for HLA-id, MUD and auto-HSCT respectively. The source of stem cells was peripheral blood stem cells for 67% of cases, and bone marrow for the remaining. The majority of grafts were non-T-cell depleted. Acute GVHD (grade I–IV) occurred in 56% of HLA-id and in 64% of MUD cases. The table shows the outcomes at two years according to the type of transplant. In conclusion, outcomes after HLA identical HSCT and MUD in adult patients with AML FAB subtype M0 in CR1 are encouraging. In comparison to allogeneic transplant cases, LFS is decreased in patients receiving an autologous transplant due to a high relapse incidence, reflecting the probable role of a graft-versus-leukemia effect in this FAB subtype. Results of HSCT in AML M0 CR1 patients Outcomes HLA-id n=150 MUD n= 30 Auto n=94 LFS: leukemia free survival; OS: overall survival; RI: relapse incidence; TRM: treatment-related mortality 2y LFS 50% 45% 33% 2y OS 58% 50% 49% 2y RI 25% 40% 57% 2y TRM 24% 14% 9%

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Leukemic Stem Cell Assessment in Remission Bone Marrow of Acute Myeloid Leukemia Patients Is a New Prognostic Parameter.

Blood ◽

10.1182/blood.v114.22.399.399 ◽

2009 ◽

Vol 114 (22) ◽

pp. 399-399 ◽

Cited By ~ 3

Author(s):

Monique Terwijn ◽

Angèle Kelder ◽

Arjo P Rutten ◽

Alexander N Snel ◽

Willemijn Scholten ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Stem Cell ◽

Myeloid Leukemia ◽

Prognostic Information ◽

Cell Compartment ◽

Hematopoietic Stem ◽

Stem Cell Compartment ◽

Acute Myeloid

Abstract Abstract 399 In acute myeloid leukemia (AML), relapses originate from the outgrowth of therapy surviving leukemic blasts know as minimal residual disease (MRD). Accumulating evidence shows that leukemia initiating cells or leukemic stem cells (LSCs) are responsible for persistence and outgrowth of AML. Monitoring LSCs during and after therapy might thus offer accurate prognostic information. However, as LSCs and hematopoietic stem cells (HSCs) both reside within the immunophenotypically defined CD34+CD38- compartment, accurate discrimination between LSCs and HSCs is required. We previously showed that within the CD34+CD38- stem cell compartment, LSCs can be discriminated from HSC by aberrant expression of markers (leukemia associated phenotype, LAP), including lineage markers like CD7, CD19 and CD56 and the novel LSC marker CLL-1 (van Rhenen, Leukemia 2007, Blood 2007). In addition, we reported that flowcytometer light scatter properties add to even better detection of LSCs, allowing LSCs detection in AML cases lacking LAP (ASH abstract 1353, 2008). Using this gating strategy, we determined LSC frequency in 64 remission bone marrow samples of CD34+ AML patients. A stem cell compartment was defined as a minimum of 5 clustered CD34+CD38- events with a minimal analyzed number of 500,000 white blood cells. After first cycle of chemotherapy, high LSC frequency (>1 × 10-3) clearly predicted adverse relapse free survival (RFS, figure 1a). LSC frequency above cut-off led to a median RFS of 5 months (n=9), while patients with LSC frequency below cut-off (n=22) showed a significantly longer median RFS of >56 months (p=0.00003). In spite of the relatively low number of patients, again a high LSC frequency (>2 × 10-4) after the second cycle and after consolidation therapy predicted worse RFS: after second cycle, median RFS was 6 months (n=9) vs. >43 months for patients with LSC frequency below cut-off (p=0.004). After consolidation, these figures were 6 months (n=7) vs. >32 months (n=6, p=0.03). Although total blast MRD (leukemic blasts as % of WBC) is known to predict survival (N.Feller et al. Leukemia 2004), monitoring LSCs as compared to total blast MRD has two major advantages: the specificity is higher (van Rhenen et al. Leukemia 2007) and well-known LSC makers like CLL-1, CD96 and CD123 can in principle be used for LSC monitoring, but not for total blast MRD detection since these markers are also expressed on normal progenitor cells. On the other hand, LSCs constitute only a small fraction of all leukemic blasts and therefore monitoring total blast MRD may have the advantage of a higher sensitivity. We thus tested the hypothesis that even more accurate prognostic information could be obtained by combining LSC frequency with total blast MRD. Total blast MRD after first cycle was predictive for survival with borderline significance (p=0.08): a cut-off of 0.3% resulted in two patient groups with median RFS of 9 months vs. >56 months. Figure 1b shows the result of the combined data of LSC and MRD frequency after first cycle therapy. We used the terms LSC+ and MRD+ for cell frequencies above cut-off and LSC- and MRD- for those below cut-off. We could clearly identify that apart from LSC+/MRD+ patients, LSC+/MRD- patients too have very poor prognosis, while MRD+/LSC- patients show an adverse prognosis as compared to LSC-/MRD- patients. These results from the first study on the in vivo fate of LSCs during and after therapy, strongly support the hypothesis that in CD34+ AML the leukemia initiating capacity originates from the CD34+CD38- population and is important for tumor survival and outgrowth. These results show that LSC frequency might be superior in predicting prognosis of AML patients in CR as compared to MRD total blast frequency, while the combination of both may offer the most optimal parameter to guide future intervention therapies. This work was supported by Netherlands Cancer Foundation KWF. Disclosures: No relevant conflicts of interest to declare.

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Prognostic Impact of Aberrant RUNX1 Expression in Patients with Acute Myeloid Leukemia Undergoing Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽

10.1182/blood.v126.23.3829.3829 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3829-3829

Author(s):

Maria Knyrim ◽

Madlen Jentzsch ◽

Marius Bill ◽

Juliane Grimm ◽

Karoline Schubert ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Normal Karyotype ◽

Myeloid Differentiation ◽

Prognostic Impact ◽

Hematopoietic Stem ◽

Travel Costs ◽

Runx1 Expression ◽

Acute Myeloid

Abstract Introduction: Despite intensive research most patients with acute myeloid leukemia (AML) still have a dismal prognosis. The transcription factor RUNX1 is a master regulator of myeloid differentiation and in AML its function is often disrupted by chromosomal translocations or mutations. These genetic alterations impact on leukemogenesis, disease progression and prognosis of AML patients. The expression of fusion proteins involving RUNX1 has been shown to exert a dominant-negative effect over wild-type RUNX1 leading to impaired myeloid differentiation and enhanced proliferation, while loss of RUNX1 may impact negatively on the frequency and self-renewal capacities of long-term hematopoietic stem cells. However, it remains unclear if differential expression of RUNX1 affects the AML phenotype. Here we analyzed the prognostic value of RUNX1 expression levels in AML patients undergoing allogeneic hematopoietic stem cell transplantation (HCT) after non-myeloablative conditioning (NMA). Patients & Methods: We analyzed 132 patients (median age 64 years [y], range 38-75y) with diagnostic bone marrow material available who received NMA-HCT (3x30mg/m2 Fludarabine on days -4 to -1 & 2Gy total body irradiation on day 0 followed by infusion of granulocyte-colony stimulating factor mobilized peripheral blood stem cells) at our institution between 2000 and 2012. Donors were human leukocyte (HLA)-matched related (15%) or HLA-matched (61%) or mismatched (24%) unrelated. 63 (48%) patients had a normal karyotype. Presence of FLT3 -ITD or FLT3 -TKD, mutational status of IDH1, IDH2, CEBPA, NPM1 and DNMT3A as well as expression levels of mir-9, mir-181a, BAALC, ERG and MN1 were determined at diagnosis. European LeukemiaNet cytogenetic classification was: 26% favorable, 27% intermediate-I, 20% intermediate-II and 27% adverse. RUNX1 expression at diagnosis was determined by qRT-PCR and normalized to 18S as internal standard. The median cut was used to define highand low RUNX1 expressers. Median follow-up was 3.9y for patients alive. Results: High RUNX1 expressers were more likely to have de novo AML (73% vs. 52%; P =.019) and higher % blasts in peripheral blood (median 38% vs. 19%; P =.004) and bone marrow (median 68% vs. 50%; P =.002) at diagnosis. Patients with high RUNX1 expression more often had IDH1 mutations by trend (P =.061) while there was no difference in IDH2 mutation frequency (P =.32). High RUNX1 expressers showed significantly higher ERG (P <.001), MN1 (P =.005) and mir-181a (P =.002) expression. High RUNX1 expression associated with longer overall (OS, P =.065) and event free survival (EFS, P =.109) by trend in the whole cohort (Figure 1 A, B). When we restricted our analysis to patients with a normal karyotype, high RUNX1 expression associated with a significantly longer OS (P =.035) and EFS (P =.041; Figure 1 C, D), while there was no prognostic impact of RUNX1 expression in patients with an abnormal karyotype (OS, P =.606 & EFS, P =.684). In multivariate analysis high RUNX1 expression independently associated with longer OS (Hazard Ratio 0.47 [95% Confidence Interval: 0.23 - 0.96]; P =.039) and longer EFS by trend (Hazard Ratio 0.57 [95% Confidence Interval: 0.29 - 1.14]; P =.113) in patients with normal karyotype. Conclusion: Our results revealed that high expression of the hematopoietic master regulator R UNX1 at diagnosis independently associated with survival in AML patients with normal karyotype receiving NMA-HCT. High RUNX1 expression associated with distinct clinical and molecular markers. Assessing pretreatment RUNX1 levels may help to refine risk stratification in AML patients undergoing NMA-HCT. Figure 1. Figure 1. Disclosures Franke: Novartis: Other: Travel Costs; MSD: Other: Travel Costs; BMS: Honoraria. Niederwieser:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

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